Protein disulfide isomerase (PDI) catalyzes the reversible formation and isomerization of disulfide bonds in proteins, and supports thrombosis. Recent reports indicate that other members of the PDI family, ERp57, ERp5, and ERp72 also contribute to thrombosis. Whether the roles of PDI, ERp57, ERp5 and ERp72 in thrombosis were distinct or redundant was unclear. We showed the aggregation defect in ERp72, PDI or ERp57-null mice was only recovered by the specific PDI that was missing. This implies that these enzymes have individual targets in the activation of the ?IIb?3 platelet integrin that supports platelet aggregation. These PDI family members contain the CGHC active-site motif that catalyzes conformational changes in proteins involved in thrombosis. This proposal focuses on two novel members of the PDI family with this motif that are found in platelets; ERp46, and a transmembrane member of the PDI family, TMX3. We now know that PDI, ERp57, ERp5 and ERp72 mediate platelet aggregation and thrombosis, and are involved in conversion of ?IIb?3 to its high affinity state. However, the actual mechanisms by which these PDIs regulate ?IIb?3 and platelet aggregation are unknown. The individual targets of each enzyme and how they function together remains an enigma. To determine the specific function of each PDI in platelets we have used a targeted knockout mice approach. The specific aims are to: 1. Characterize the role of ERp46 in thrombus formation and platelet function; 2. Characterize the role of TMX3 in thrombus formation and platelet function; and 3. Characterize the cysteine/disulfide targets and mechanism of activation of ?IIb?3 by PDI, ERp57, ERp72, ERp46 and TMX3. A principal technique used will be the laser-induced injury model of thrombosis. To determine the actual mechanisms by which multiple members of the PDI family work together we will employ a thiol labeling strategy with mass spectrometry identification of the labeled thiols. This will begin to unravel the mechanisms by which these enzymes work individually, and how they work together as a network. Elucidation of the extracellular redox network required for the final steps in the activation of ?IIb?3 represents a significant aspect of platelet function and thrombus formation, and can be a model for activation of other integrins. Defining the specific mechanisms could also lead to novel types of inhibitors that dually regulate platelets and coagulation. Since platelets are involved in a variety of disease states, our findings will likely have broader implications for basic understanding of other disease conditions, and possible therapeutic approaches for these conditions.